Ionizing radiation induces DNA double stand breaks, which activates a signal cascade initiated by ATM/ATR kinases. The damage signals stop the cell cycle progression and allow enough time for DNA damage repair. Here, we have identified an important regulator, RNF8 (Ring Finger protein 8) that participates in the activation of DNA damage response. Following DNA damage, the FHA domain of RNF8 recognizes phosphorylated MDC1, a binding partner of histone H2AX in DNA damage response, which targets RNF8 to DNA damage sites. Histone H2AX is a scaffold protein that retains DNA damage response proteins at the DNA damage sites. Since RNF8 is an E3 ubiquitin ligase, we have found that RNF8 controls histone H2AX ubiquitination following DNA damage. RNF8-dependent H2AX ubiquitination may regulate downstream 53BP1 (p53 Binding Protein 1) and BRCA1 (Breast Cancer Susceptibility Gene 1) translocation to DNA damage sites. Both BRCA1 and 53BP1 are not only downstream targets of ATM/ATR kinases, but also important tumor suppressors. Moreover, we have generated RNF8 knock-out mice. The RNF8 null mice are viable. But male RNF8 -/- mice are sterile. RNF8 -/- mice are hypersensitive to ionizing radiation. These phenotypes are very similar to those of histone H2AX and MDC1 knock-out mice. Thus, based on our preliminary studies, we hypothesize that RNF8 plays a major role in activation of DNA damage response and participates in tumor prevention. We propose following experiments to examine our hypotheses. Aim1: To investigate the role of RNF8-dependent histone ubiquitination in DNA damage response. Aim2: To study the role of RNF8 in histone methylation in response to DNA damage. Aim3: To examine the role of RNF8 in tumor suppression. In summary, our proposed studies will not only provide insights into the mechanisms of the activation of DNA damage response, but also contribute to our understanding of the maintenance of genomic integrity and tumor prevention.